Pharmaceutical composition and method for regenerating myofibers in the treatment of muscle injuries

ABSTRACT

A pharmaceutical composition and method for regenerating cardiomyocytes in treating or repairing heart muscle damages or injuries caused by an ischemic disease. The pharmaceutical composition contains an active ingredient compound with a backbone structure of Formula (I). The active ingredient compound is capable of (a) increasing viability of myogenic precursor cells to enable said precursor cells to survive through an absolute ischemic period; (b) reconstituting a damaged blood supply network in said heart region where said injured muscle is located; and (c) enhancing cardiomyogenic differentiation efficiency of said precursor cells down cardiac linage, said steps being performed simultaneously or in any particular order.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/843,898, filed Sep. 2, 2015, which is a divisional of U.S. patentapplication Ser. No. 11/722,915, filed Jun. 27, 2007, now U.S. Pat. No.9,155,744, issued Oct. 13, 2015, which is a §371 national stage patentapplication of International Patent Application No. PCT/CN2006/002885,filed Oct. 27, 2006, and which claims priority to U.S. ProvisionalApplication No. 60/791,462, filed Apr. 13, 2006, and to InternationalPatent Application No. PCT/IB2005/003202, filed Oct. 27, 2005 and toInternational Patent Application No. PCT/IB2005/003191, filed Oct. 27,2005, the contents of all of which are hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to a pharmaceutical composition and a method ofregenerating myocytes and myocardium for treating muscle damages.Particularly, it relates to a pharmaceutical composition and method forregenerating cardiomyocytes in treating or repairing heart muscledamages or injuries caused by an ischemic disease.

BACKGROUND OF THE INVENTION

Myocardial infarction (MI), or heart attack, is a disease due tointerruption of the blood supply to a part of the heart, causing damageor death of heart muscle cells. It is the leading cause of death forboth men and women over the world. Following myocardial infarction,there does not seem to be any natural occurring repairing processcapable of generating new cardiomyocytes to replace the lost musclecells. Instead, scar tissues may replace the necrosed myocardium,causing further deterioration in cardiac function.

Therapeutic replacement of the necrosed heart tissue with newlyregenerated functional cardiac myocytes is a treatment ideal that untilrecently has been unrealistic, because cardiac myocytes were consideredto be terminally differentiated, or in other words, the heart is apostmitotic nonregenerating organ. This dogma, however, has recentlybeen challenged by Beltrami et al, and others, who reported that apopulation of resident myocytes within the myocardia can and doreplicate after infarction. In order to promote and improve the repairfor infarcted myocardia, transplantation of cardiomyocytes or skeletalmyoblasts has been attempted, but has not been very successful inreconstituting functional myocardia and coronary vessels.Transplantation of adult bone marrow-derived mesenchymal stem cells(MSCs) for cardiac repair following myocardial infarction has resultedin some angiogenesis and myogenesis, but the location of the newlyregenerated cardiac myocytes appeared mostly along the border zone wherethe blood supply is relatively less affected ¹⁻³.

Because acute myocardial infarction (MI) brings rapid damages or deathto myocytes (heart muscle cells), vascular structures and nonvascularcomponents in the supplied region of the ventricle, regeneration of newcardiac myocytes to replace the infarcted myocardia (heart musculartissues) in the central infarcted zone (the absolute ischemic region)through a sub-population of cardiac myocyte growth ⁴⁻⁸ ortransplantation of MSCs ¹⁻³ alone appears to be impossible without earlyreestablishment of the blood supply network locally. This probablyexplains why regeneration of cardiac myocytes following MSCstransplantation alone occurred mostly along the border zone adjacent tothe infarct where the blood supply is largely maintained ¹⁻¹¹.Therefore, the loss of myocardia, arterioles and capillaries in thecentral infarct area appeared to be irreversible, eventually leading toscar formation.

A more recent study ¹² reported that heart transplantation of MSCpre-modified with exogenous Akt in vitro produced a better result.Nonetheless, the regenerated cardiac myocytes could only infiltrate fromthe border zone into the scarred area, indicating that overexpression ofexogenous Akt, although enhancing the survival potential of thetransplanted MSCs, itself is insufficient to enable them to survive incentral ischemic regions. Furthermore, even in the less-ischemic borderzones, it was noted that the MSCs-derived regenerating cardiac myocyteswere scattered and seemed to have difficulty to cluster and formregenerating myocardia. This is probably due to poor cardiomyogenicdifferentiation efficiency of the survived transplanted MSCs. Theknowledge that natural cardiomyocyte reproduction, includingdifferentiation of residential progenitor myocytes or stem cellsrecruited from other sources, such as from endothelial cells or a nichein the bone marrow is insufficient to balance cardiomyocyte deathoccurred in acute or chronically damaged heart, has damped theenthusiasm of the researchers who thought myocardial regeneration wouldrepresent a promising method of treatment against heart diseases.

The prior art seems to teach that there are three major requirementscritical for regenerating functional myocytes in the entire areas ofinfarcted myocardia: 1) increased viability of the transplanted cells sothat they may survive through the absolute ischemic period, that is, theperiod from injection of the donor cells to formation of new vessels; 2)early reconstitution of the damaged blood supply network in theinfarcted myocardia to sustain the survival and efficient trafficking ofthe transplanted cells and maintain oxygenation and nutrient delivery;and 3) enhanced cardiomyogenic differentiation efficiency of thetransplanted cells to enable more survived donor cells to differentiatedown cardiac linage.

Therefore, to realize the therapeutic ideal of replacing necrosed hearttissues with newly regenerated functional cardiac myocytes, there is aneed for new therapeutic approaches, for example, an approach usingchemical compounds possessing biological properties that sufficientlysatisfy the aforementioned three requirements in order to serve thetherapeutic needs for treating myocardial infarction.

SUMMARY OF THE INVENTION

As one object of the present invention, there is provided apharmaceutical composition comprising a compound selected from the groupof chemical compounds sharing a common backbone structure of formula(I). The compounds have potent beneficial therapeutic effects not onlyon the survival potential and cardiogenic differentiation efficiency ofMSCs ex vivo, but also on repairing of MI in vivo. These compoundsthemselves are known in the art but they are never known as possessingthe above biological activities and therapeutic effects. They may beisolated from natural resources, particularly from plants or they mayalso be obtained through total or semi-chemical syntheses, with existingor future developed synthetic techniques. The backbone structure itselfpossesses the aforementioned myogenic effects and various variants canbe made from the backbone structure through substitution of one or morehydrogen atoms at various positions. These variants share the commonbackbone skeleton and the myogenic effects. Of course, they may vary inmyogenic potency.

The backbone structure of Formula (I) may have one or more substituentsattached. A substituent is an atom or group of atoms substituted inplace of the hydrogen atom. The substitution can be achieved by meansknown in the field of organic chemistry. For example, through a properdesign, high through-put combinatorial synthesis is capable of producinga large library of variants or derivatives with various substituentsattached to various positions of a backbone structure. The variants orderivatives of formula (I) may then be selected based on an activitytest on mesenchymal stem cells (MSCs), which can quickly determinewhether a particular variant could enhance proliferation and cardiogenicdifferentiation of the cultured MSCs. As used in this application, theterm “the compound of formula (I)” encompasses the backbone compounditself and its substituted variants with similar biological activities.

Examples of these variants are presented in the following, all of whichpossess similar effects in terms of regenerating functional myocytes asthe backbone structure (i.e., the base compound itself):

Furthermore, as a therapeutic agent, the compound of formula (I) may bein a form of “functional derivatives” as defined below.

It is contemplated, as a person with ordinary skill in the art wouldcontemplate, that the above compounds may be made in various possibleracemic, enantiomeric or diastereoisomeric isomer forms, may form saltswith mineral and organic acids, and may also form derivatives such asN-oxides, prodrugs, bioisosteres. “Prodrug” means an inactive form ofthe compound due to the attachment of one or more specialized protectivegroups used in a transient manner to alter or to eliminate undesirableproperties in the parent molecule, which is metabolized or convertedinto the active compound inside the body (in vivo) once administered.“Bioisostere” means a compound resulting from the exchange of an atom orof a group of atoms with another, broadly similar, atom or group ofatoms. The objective of a bioisosteric replacement is to create a newcompound with similar biological properties to the parent compound. Thebioisosteric replacement may be physicochemically or topologicallybased. Making suitable prodrugs, bioisosteres, N-oxides,pharmaceutically acceptable salts or various isomers from a knowncompound (such as those disclosed in this specification) are within theordinary skill of the ait. Therefore, the present invention contemplatesall suitable isomer forms, salts and derivatives of the above disclosedcompounds.

As used in this application, the term “functional derivative” means aprodrug, bioisostere, N-oxide, pharmaceutically acceptable salt orvarious isomer from the above-disclosed specific compound, which may beadvantageous in one or more aspects compared with the parent compound.Making functional derivatives may be laborious, but some of thetechnologies involved are well known in the art. Various high-throughputchemical synthetic methods are available. For example, combinatorialchemistry has resulted in the rapid expansion of compound libraries,which when coupled with various highly efficient bio-screeningtechnologies can lead to efficient discovering and isolating usefulfunctional derivatives.

The pharmaceutical composition of the present invention is useful fortreating myocardial injuries or necrosis caused by a disease,particularly MI, through regenerating heart tissues. The pharmaceuticalcomposition may be formulated by conventional means known to peopleskilled in the art into a suitable dosage form, such as tablet, capsule,injection, solution, suspension, powder, syrup, etc, and be administeredto a mammalian subject having myocardial injuries or necrosis. Theformulation techniques are not part of the present invention and thusare not limitations to the scope of the present invention.

The pharmaceutical composition of the present invention may beformulated in a way suitable for oral administration, systemicinjection, and direct local injection in the heart or implantation in abody part for long-term slow-releasing.

In another aspect, present invention provide a method for treating orameliorating a pathological condition in a mammal, where thepathological condition, as judged by people skilled in medicine, can bealleviated, treated or cured by regenerating functional cardiomyocytesand where the method comprises administering to the mammal with thepathological condition a therapeutically effective amount of a compoundof formula (I) and or its functional derivatives.

In another aspect, the present invention provides a method ofregenerating functional cardiomyocytes in a mammal who needs to replacedead or damaged heart tissues caused by a heart disease, such as,myocardial infarction (MI). This is a cell-transplantation basedtherapeutic approach, involving the steps of: (a) obtaining stem cells,such as MSCs; (b) contacting the stem cells with a compound of theformula (I) or their functional derivatives to activate the pathways ofcardiogenic differentiation prior to transplantation and (c) thentransplanting the activated cells into the infarcted heart tissues ofthe mammal. This therapeutic approach is capable of achieving thefollowing goals: 1) enhanced survival potential of the transplantedcells; 2) early reconstitution of blood supply network, and 3) enhancedcardiomyogenic differentiation efficiency of the transplanted cells byex vivo activation of MSCs forming cardiogenic progenitors prior totransplantation.

In another aspect, the present invention provides a method for treatingischemic heart diseases, particularly MI in mammals, which comprisingthe steps: (a) culturing MSCs or endothelial cells with a compound offormula (I) or their functional derivatives, (b) gathering theconditioned medium of the treated cells, which contains secretaryproteins that are active in driving heart infarction repair orcardiogenic differentiation of MSCs, and (c) administering or deliveringthe conditioned medium to the heart tissue in the infarct area.

In still another aspect, the present invention provides a researchreagent for scientific research on cardiogenic transdifferentiation ofstem cells, such as MSCs. The reagent comprises one or more compounds offormula (I) or their functional derivatives. It may be in a solid formor a liquid form. For example, it may be a solution of DMSO.

The various features of novelty which characterize the invention arepointed out with particularity in the claims annexed to and forming apart of this disclosure. For a better understanding of the invention,its operating advantages, and specific objects attained by its use,reference should be made to the drawings and the following descriptionin which there are illustrated and described preferred embodiments ofthe invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 outlines the process of isolating Niga-ichigoside Fl (referred toas “CMF”) from the plant of Geum Japonicum as an example of making thecompound of the present invention.

FIG. 2A shows the effects of CMF on cardiogenic differentiation of theMSCs and up-regulation of phospho-Akt1 expression ex vivo by antibodystaining. FIG. 2B shows Western bloat analysis to confirm AKt1upregulation.

FIG. 3 shows the therapeutic effect of a treatment based ontransplantation of CMF-pretreated MSCs.

FIG. 4 shows distribution ejection fraction (EF) and fractionalshortening (FS) after 2 days and 2 weeks cell transplantation in threegroups of rats (A: normal group; B: MI group transplanted withCMF-treated MSCs; C: MI group transplanted with MSCs not treated withCMF).

FIG. 5 shows the therapeutic effects of CMF on myocardial infarction(Ml) animal model.

FIG. 6A shows the enhanced proliferation of cultured MSCs and myocardialregeneration induced by conditioned medium. FIG. 6B shows the effect oncardiomyocyte regeneration after injection of conditioned medium intoischemic regions in an MI model.

FIG. 7 shows the cellular source for CMF-induced myocardial regenerationin animal MI model.

DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS I. Experiment Procedures

All protocols used in the present invention conformed to the Guide forthe Care and Use of Laboratory Animals published by the U.S. NationalInstitutes of Health, and were approved by the Animal ExperimentalEthical Committee of The Chinese University of Hong Kong.

For the following discussion, CMF refers to the base compound (orbackbone compound) of the present invention. Its chemical structure isdefined by formula (I) shown in the above.

Obtaining Compounds of the Present Invention: The compounds can beprepared from plants, although it may possible to make it throughchemical synthesis.

As an example for illustrating the process of preparing the compoundsfrom natural resources, the following provides details involved in CMF'sisolation and purification from one plant species, Geum Japonicum. Otherplants that may contain CMF or variants include, for example, Acaenapinnatifida R. et P., Agrimonia pilosa Ledeb, Asparagus filicinus,Ardisia japonica, Campsis grandiflora, Campylotropis hirtella (Franch.Schindl.), Caulis Sargentodoxae, Cedrela sinensis, Chaenomeles sinensisKOEHNE, Debregeasia salicifolia, Eriobotrya japonica calli, Eriobotryajaponica LINDL. (Rosaceae), Goreishi, Leucoseptrum stellipillum,Ludwigia octovalvis, Perilla frutescens, Perilla frutescens (L.) Britt.(Lamiaceae), Physocarpus intermedius Potentilla multifida L., Poteriumancistroides, Pourouma guianensis (Moraceae), Rhaponticum uniflorum,Rosa bella Rehd. et Wils., Rosa laevigata Michx, Rosa rugosa, Rubusalceaefolius Poir, Rubus allegheniensis, Rubus coreanus, Rubusimperialis, Rubus imperialis Chum. Sehl. (Rosaceae), Rubus sieboldii,Rumex japonicus, Salvia trijuga Diels, Strasburgeria robusta, Strawberrycv. Houkouwase, Tiarella polyphylla, Vochysia pacifica Cuatrec,Zanthoxylum piperitum, etc.

Isolation of cardiomyogenic factor (CMF) from Geumjaponicum: Referringto FIG. 1, the plant of Geum japonicum collected from Guizhou Provinceof China in August was dried (IOkg) and percolated with 70% ethanol(IOOL) at room temperature for 3 days twice. The extract was combinedand spray-dried to yield a solid residue (1 kg). The solid residue wassuspended in 10 liter H₂O and successively partitioned with chloroform(10 L) twice, then n-butanol (10 L) twice to produce the correspondingfractions. The n-butanol (GJ-B) soluble fraction was filtered and driedby spray drying to yield a powder fraction, which was confirmed fortheir specific ability to stimulate cardiogenic differentiation of MSCsin cell culture in a way described below. It was shown that n-butanolsoluble fraction (GJ-B) could enhance the proliferation and cardiogenicdifferentiation of the cultured MSCs in cell culture systems. The GJ-Bfraction was then applied on a column of Sephadex LH-20 equilibratedwith 10% methanol and eluted with increasing concentration of methanolin water, resolving 7 fractions, GJ-B-1 to GJ-B-7. All the elutedfractions were tested for their activity with MSC culture systems.Activity test demonstrated that fraction 6 was most active in enhancingthe cardiogenic differentiation of cultured MSCs. From GJ-B-6, a pureactive compound was further isolated, which is referred to as CMFthrough this disclosure. CMF's structure was determined by NMR analysisand comparison with literature, and shown to be of formula (I).

Preparation of MSCs for transplantation: The MSCs were cultured with CMF(10 μg/ml in growth medium) for 6 days. In parallel, the control MSCswere cultured in growth medium containing equivalent volume of 5% DMSO.On day 2, expression of endogenous phospho-Akt1 was assessed byimmunocytochemistry and Western blot. On day 4, myogenic differentiationwas assessed by immunocytochemistry and Western blot against MEF2, whichwere further confirmed by immunocytochemistry and Western blot with anantibody specific to MHC on day 6. On day 3, both the CMF-pretreatedMSCs and the control MSCs were labeled with CM-DiI in culture and madeready for transplantation.

Preparation of bone marrow mesenchymal stem cells: The tibias/femurbones were removed from Sprague-Dawley (SD) rats and the bone marrow(BM) was flushed out of the bones with IMDM culture medium containing10% heat inactivated FBS (GIBCO) and 1% penicillin/streptomycin. The BMwas thoroughly mixed and centrifuged at 1500 rpm for 5 minutes. The cellpellet was suspended in 5 ml growth medium. The cell suspension wascarefully put on 5 ml Ficoll solution and centrifuged at 200 rpm for 30min. The second layer, which contains BM cells was transferred into atube and washed twice with PBS to remove Ficoll (1200 rpm for 5minutes). The cell pellet was resuspended in IMDM culture mediumcontaining 10% heat inactivated FBS (GIBCO) and 1%penicillin/streptomycin antibiotic mixture. After 24 hours culture in a37° C. incubator with 5% CO2, the non-adherent cells are discarded andthe adherent cells are cultured by changing medium once every 3 days andthe cells became nearly confluent after 14 days of culture. This was theBM cells, referred to as MSCs in the following, which were used for invitro and in vivo studies conducted in the present disclosure.

Western blot analysis: Whole cell extracts of the CMF-treated cells orcontrol cells were prepared by lysing the cells with 3 times packed cellvolume of lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCI, 1 mM EDTA, I mMEGTA, 1% Nonidet P-40, 10% glycerol, 200 mM NaF, 20 mM sodiumpyrophosphate, 10 mg/ml leupeptin, 10 mg/ml aprotinin, 200 mMphenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate) on ice for30 minutes. Protein yield was quantified by Bio-Rad DC protein assay kit(Bio-Rad). Equal amounts (30 μg) of total protein were size-fractionatedby SDS-PAGE and transferred to PVDM membranes (Millipor). The blots wereblocked with phosphate-buffered saline plus 0.1% (vol/vol) Tween 20(PBST) containing 5% (wt/vol) milk powder (PBSTM) for 30 minutes at roomtemperature and probed for 60 minutes with specific primary antibod iesagainst rat phospho-Akt1 (mouse) or rat MHC (mouse, Sigma-Aldrich),diluted 1:1000 in PBSTM. After washing extensively in PBST, the blotswere probed by horseradish peroxidase-coupled anti-mouse IgG (AmershamBiosciences) (1/1000 dilution in PBSTM, 60 min), extensively washed withPBST, and developed by chemiluminescence.

Transplantation of the CMF-pretreated MSCs to the heart tissue: TheSprague-Dawley (SD) rats were used and all animal procedures wereapproved by the University Animal Committee on Animal Welfare. Each ratwas anesthetized with intraperitoneal pentobarbital (50 mg/kg),intubated, and mechanically ventilated with room air using a Harvardventilator (model 683). After a left thoracotomy, myocardial infarctionwas induced by permanent ligation of left anterior descending (LAD)coronary artery. The 5×10⁵ DiI labeled CMF-pretreated MSCs (32 rats)suspended in saline were injected into three sites of the distalmyocardia (the ischemic region) of the ligated artery immediate afterthe ligation respectively (test group). The control rats were injectedwith an equivalent amount of DiI labeled non-treated control MSCs (32rats) suspended in saline at the same location and timing. For shamischemia (32 rats), thoracotomy was performed without LAD ligation.Sixteen rats subject to no-treatment were set as normal control.

Half of the experimental rats from different groups were sacrificedaccording to experimental plan on day 7 and day 14 post-infarction afterassessment of their heart function by echocardiography measurements. Thehearts of the sacrificed rats were removed, washed with PBS andphotographed respectively. All the specimens harvested were paraffinembedded and sectioned for tracing the signals of Oil and examination ofrevascularization, infarct size and regeneration of myocardia. If theregenerating cells were DiI positive, further MHC immonohistochemicalstaining was performed to confirm their cardiomyogenic differentiation.

Colocalization of the DiI label and cardiac-specific marker expressionwere examined with a confocal microscope (ZEISS, LSM 510 META). Briefly,the sections were immunohistochemically stained with rat-specifictroponin I antibodies. The confirmation of cardiomyogenicdifferentiation of the Oil labeled transplanted MSCs formingregenerating myocardia was carried out by merging the DH-positive cells,indicating their donor cell origin, with the specific positive stainingof cardiac terminal differentiation marker-troponin I using confocalmicroscopic examination, implying their cadiomyogenic differentiation ofthese transplanted cells.

CMF direct treatment in MI model: Thirty-two SD rats were randomlydivided into four groups: normal group, sham group, CMF-treated groupand non-treated control group (8 rats each). Rats were anesthetized withintraperitoneal pentobarbital (50 mg/kg), intubated, and mechanicallyventilated with room air using a Harvard ventilator (model 683). After aleft thoracotomy, myocardial infarction was induced by permanentligation of left anterior descending (LAD) coronary artery. CMF in 5%DMSO (0.1ml, containing 0.1 mg CMF) was injected into the distalmyocardium (the ischemic region) of the ligated artery in 8 ratsimmediate after the ligation (CMF-treated group). Another 8 rats wereinjected with an equivalent amount of 5% DMSO at the same location andtiming as non-treated control group. For sham ischemia, thoracotomy wasperformed on 8 rats without LAD ligation. Further 8 rats without anytreatment were set as normal group.

Conditioned medium containing secretary proteins from MSCs or othercells induced by CMF: The MSCs were treated with 10 μg/ml CMF for 24hours to activate/upregulate gene expressions and then washed thoroughlyto remove residue of CMF. Then 5 ml of fresh growth medium was added tothe culture and collected after another 3 days of culturing. Thecollected medium was referred to as conditioned medium. The 5 ml ofconditioned medium was condensed to a volume of 1 ml, and was used as atreatment agent in the heart infarction animal model as described above.Briefly, after a left thoracotomy and ligation of LAD, 0.2 ml of theconditioned medium was injected immediately into the distal part ofligation. Fresh growth medium was used as control.

Bone marrow replacement with DU labeled MSCs: Sixteen 5-week old SD ratswere used for bone marrow transplantation. Recipient rats were irritatedby 9.5 Gy of gamma irradiation from a 137Cs source (Elite Grammacell1000) at a dosage of 1.140 Gy/min to completely destroy the bone marrowderived stem cells of the rat. Oil-labeled MSCs (2×10⁸ cells suspendedin 0.3 ml PBS) were then injected through tail vein within 2 hours afterirritation using a 27-gauge needle. One week after irritation andtransplantation, the rats with Oil-labeled bone marrow were divided intotwo groups: one to be treated directly with CMF and the other as controlwithout treatment. Heart infarction surgery and treatment scheme wereperformed as described above. The experiment was terminated on day 14post surgery and treatment for further assessment. Heart specimens ofthe sacrificed rats were obtained. All the specimens were traced for DiIpositive cells and their cardiomyogenic differentiation byimmunohistochemical staining with specific antibodies for heart typetroponin I(Santa Cruz) and PCNA (Dako). Specific secondary antibodyconjugated with alkaline phosphatase (Santa Cruz) was used to visualizethe positively stained cells. DiI positive signal was observed with afluorescence microscope (Laica)

Estimation of infarct size: Left ventricles from experimental ratssacrificed on day 14 were removed and sliced from apex to base in 3transverse slices. The slices were fixed in formalin and embedded inparaffin. Sections (20 μm thickness) of the left ventricle were stainedwith Masson's trichrome, which labels collagen blue and myocardium red.These sections were digitized and all blue staining was quantifiedmorphometrically. The volume of infarct (mm³ of a particular section wascalculated based on the thickness of the slice. The volumes ofinfarcted·tissue for all sections were added to yield the total volumeof the infarct for each particular heart. All studies were performed bya blinded pathologist.

Angiogenic assessment in infarct region: Vascular density was determinedon day 7 postinfarction from histology sections by counting the numberof vessels within the infarct area using a light microscope under highpower field (HPF) (×400). Six random and non-overlapping HPFs within theinfarct filed were used for counting all newly formed vessels in eachsection of all experimental hearts. The number of vessels in each HPF isaveraged and expressed as the number of vessels per HPF.

Assessment of regenerating cardiac myocytes and myocardia: The sectionsfrom both CMF pretreated MSCs-transplanted and non-treatedMSCs-transplanted groups on day 7 post-ligation were stained with Ki67or myosin heavy chain (MHC) antibodies to identify the regeneratingmyocardia. Specific secondary antibody conjugated with alkalinephosphatase was used to visualize the positive stains. Briefly,paraffin-embedded sections were microwaved in a 0.1 M EDTA buffer andstained with a polyclonal rabbit antibody with specificity against ratKi67 at 1:3,000 dilution (Sant Cruz Biotechnology) and incubatedovernight at 4° C. After they were washed, the sections were incubatedwith a goat anti-rabbit IgG secondary antibody conjugated with alkalinephosphatase at 1:200 dilution (Sigma) for 30 min, and the positivenuclei were visualized as dark blue with a5-bromo-4-chloro-3-indolylphosphate-p-toluidine-nitro blue tetrazoliumsubstrate kit (Dako). The immediate neighbor sections from correspondingparaffin tissue block were incubated overnight at 4° C. in a 1:50dilution of rabbit anti-rat MHC (MF20, Developmental Hybridoma Bank,University of Iowa) antibodies, and further incubated for 30 minutes atroom temperature in a 1:100 dilution of peroxidase-conjugated goatanti-rabbit IgG (Sigma). After incubation with 1 mg/ml3,3′-diaminobenzidine (DAB; plus 0.02% H₂O₂) the slides wereinvestigated by microscopic analysis. The regenerating myocardium areawas delineated in the projected field by a grid containing 42 samplingpoints. Approximately, 30-60 calculating points along the border of aparticular regenerating myocardium were selected in each section.

This grid defined an uncompressed tissue area of 62,500 μm², which wasused to measure the selected 30-60 calculating points in each section.The shapes and volumes of regenerating myocardia in the central area ofinfarct were determined by measuring in each section (50 μm apart) ofapproximately 70 sections the shapes and areas occupied by theregenerating myocardia and section thickness. Integration andcalculation with these variables produced a stereo-structure and yieldsthe volume of a particular regenerating myocardium in the central areaof the infarct in each section. Values and stereo structure of allsections of a particular tissue block were added and computed to obtainthe total volume and the full stereo-structure of the regeneratingmyocardia.

Echocardiography assessment of myocardial function: Echocardiographicstudies were performed using a Sequoia C256 System (Siemens Medical)with a 15-MHz linear array transducer. The chest of experimental ratswas shaved, the animal was situated in the supine position on a warmingpad, ECG limb electrodes were placed, and echocardiography was recordedunder controlled anesthesia. Each experimental rat received a baselineechocardiography before the experimental procedure. Two-dimensionalguided M-mode and two-dimensional (2D) echocardiography images wererecorded from parasternal long- & short-axis views. Left ventricular(LV) end-systolic and end-diastolic dimensions, as well as systolic anddiastolic wall thickness were measured from the M-mode tracings by usingthe leading-edge convention of the American Society of Echocardiography.LV end-diastolic (LVDA) and end-systolic (LVSA) areas were planimeteredfrom the parasternal long axis and LV end-diastolic and end-systolicvolumes (LVEDV and LVESV) were calculated by the M-mode method. LVejection fraction (LVEF) and fractional shortening (FS) were derivedfrom LV cross-sectional area in 2D short axis view:EF=[(LVEDV−LVESV)/LVEDV]×100% and FS=[(LVDA−LVSA)/LVDA]×100%. Standardformulae were used for echocardiographic calculations. All data wereanalyzed offline with software resident on the ultrasound system at theend of the study. All measured and calculated indexes were presented asthe average of three to five consecutive measurements.

Statistics: All morphometric data are collected blindly, and the code isbroken at the end of the experiment. Results are presented as mean±SDcomputed from the average measurements obtained from each heart.Statistical significance for comparison between two measurements isdetermined using the unpaired two-tailed Student's t test. Values ofP<0.05 are considered to be significant.

II. CMF-Induced Increased Survival Potential and CardiogenicDifferentiation of MSCs Ex Vivo

Referring to FIG. 2, following two days treatment with CMF (10 μg/ml) inthe culture, the expression of phospho-Akt 1 was significantlyup-regulated compared with the untreated control as demonstrated byimmunocytochemical staining of the cells with antibodies specific tophospho-Akt1, positive cells being stained red mainly in the cytoplasm(FIG. 2 a: 1). Western blot confirmed that the increased expression ofphospho-Akt1 up to 3-4 folds over untreated cells (FIG. 2b : SA). Amongthe phospho-Akt1 up-regulated MSCs, more than 90% of them when culturedfor 2 additional days became positively stained with the antibodyspecific to myocyte enhancer factor 2 (MEF2), one of the earliestmarkers for the cardiogenic lineage, positive cells being stained orangein the nuclei (FIG. 2 a: 2) and confirmed by Western blot (FIG. 2 b:6A), indicating their commitment to cardiogenic differentiation. It wasnoted that the cultured MSCs were not all positively stained byanti-MEF2 antibody (FIG. 2 a: 2), as indicated by the blue nucleuspossibly because not all cultured MSCs were converted down thecardiogenic differentiation pathway by CMF or because some minorimpurities existed in the preparation of MSCs. Similarly, most of thecultured MSCs were positively stained with the antibody specific toheart type myosin heavy chain, positive cells being stained red in thecytoplasm (FIG. 2 a: 3) and confirmed by Western blot (FIG. 2 b: 7A)upon 6 days culture in the presence of CMF, while control cells werenegative stained by all three specific antibodies (FIGS. 2 a: 4 & 2 b:Bs). The sequential induction of MEF2 and MHC expressions confirmed theCMF-induced cardiogenic differentiation development of MSCs ex vivo. InFIG. 2b , A stands for CMF-treated sample while B for untreated control.

III. Therapeutic Effect via Transplantation of MSCs Pretreated with CMF

To determine whether the increased survival potential and cardiogenicdifferentiation efficiency showed ex vivo in MSCs treated with CMF priorto transplantation would bring about significant improvement inrepairing of MI in vivo, or in other words, whether CMF's ex vivoeffects have any therapeutic values, cell transplantation experimentswith MI animal model were performed, where MSCs pre-treated with CMFwere implanted in the areas of infarct. The homing, survival,proliferation, cardiomyogenic differentiation and maturation of thetransplanted cells were traced by the positive signals of eitherDil-florescence and by immunohistochemical staining for Ki67 and MHC insections, which were from the hearts on day 7 and day 14 post infarctionand cell transplantation. As shown in FIG. 3, DiI positive cells on day7 (FIG. 3: 1) with the characteristic phenotype of a cardiac myocytewere observed in the whole area of the infarct in the test myocardiumgroup, indicating their donor cell origin and whole infarct zonedistribution. In control group (untreated with CMF), only scattered DiIsignals around the infarct border could be seen (FIG. 3: 2). Thecolocalization of DiI signals (red) and cardiac specific troponin Iexpression (green) was observed (FIG. 3: 3-5) in the whole infarct zoneby confocal microscopy. The merged image of Dil-positive (red) andcardiac specific marker troponin I expression (green) resulted inyellow-red-green overlapping colors in the same cells, thus confirmingthe in vivo cardiomyogenic differentiation and maturation of thetransplanted MSCs pretreated ex vivo with CMF. It was also noted that afew troponin I positive cells (green) were not Dil positive (FIGS. 3: 3& 5), probably because some regenerating myocytes were not derived fromthe transplanted Dil-labeled-MSCs. Similarly, a few Dil positive cellsshown in the light blue circles (FIGS. 3: 4 & 5), were negative introponin I immunostaining, indicating a small fraction of thetransplanted cells did not commit cardiogenic differentiation in vivo,or impurities contained in the preparation of MSCs.

Formation of new vessels could be detected as early as 12 hours aftertransplantation and many more newly formed vessels and capillariesfilled with blood cells were observed in the whole infarct areas in thetest group in 24 hours (before any regenerating cardiac myocytes couldbe seen) and in 7 days post infarction (FIG. 3: 1, yellow circles). Thedensity of the newly formed vessels in the infarct area of the CMFpretreated MSCs transplanted myocardia was on average 8±2 per high powerfield (40×) (HPF) on day 7. However, the new vessels were notDH-positive, indicating that the cellular source of the vessels may notbe derived from the donor cells. It is contemplated that the donor MSCsmay be activated by CMF-pretreatment to·stimulate and upregulateangiogenesis specific signaling pathways that induce the expression ofcertain angiogenic factors, which directly enhances the process of earlyrevascularization in infarcted myocardia. By contrast, approximately 3±2vessels per HPF were observed in the infarcted myocardia ofnon-pretreated MSCs transplanted controls on day 7 (FIG. 3: 2, yellowcircles).

As shown in FIG. 3, a large number of donor cell derived myocytes wereclustered and organized into myocardial-like tissue in the infarct area,which were positively stained by antibodies specific to MHC (FIG. 3: 6,blue circles) and Ki67 (FIG. 3: 7, blue circles), indicating that thesetransplanted CMF-pretreated MSCs retained the division ability andcommitted cardiomyogenic differentiation after transplantation in vivo.Under a high power field, these myocardial-like tissues showed thetypical morphology of myocardium, except the size was smaller thanundamaged existing myocytes (FIG. 3: 9, blue circles). These highlyorganized regenerating myocardium-like tissues occupied averagely 70±8%of the total infarct volume on day 7 and replaced the infarctedmyocardia by 80±8.5% on average on day 14 post-infarction in the testmyocardium group (FIG. 3: 8, R, regenerated cardiac myocytes; N,preexisting normal cardiac myocytes). This replacement of the infarctedheart tissue was accompanied by significant functional improvement, asdemonstrated in the echocardiography measurements (FIG. 4 & Table 1). Incomparison with the non-pretreated MSCs transplanted MI group on day 2and 14 post infarction, ejection fraction (EF) of thepretreated-MSCs-transplanted MI hearts was significantly higher(59.79±2.33 vs 52.1±2.54, P=0.03) on day 2, and markedly increased(67.13±2.53 vs 53.3±2.31, P=0.001) on day 14. Similarly, fractionshortening (FS) of the transplanted MI heart were significantly higher(29.43±1.35 vs 24.07±1.47, P=0.01) on day 2 and was significantlyincreased (31.72±2.57 vs 23.49±1.99,P=0.002) on day 14. The significantimprovements in EF and FS are a solidreflection of the functionalrecovery of the cardiac myocytes (Table 1).

TABLE 1 The distribution of ejection fraction (EF) and fractionalshortening (FS). (mean ±_SE): EF (%) FS (%) 2 14 days 2 14 days 

71.03 ± 4.05 68.24 ± 4.79 35.65 ± 3.99 34.02 ± 3.27 CMF- 59.79± 67.13±29.43± 31.72± pretreated MSC control 52.1± 53.3± 24.07± 23.49 ± 1.99 §,Sixteen rats for normal group and 32 rats for sham operated,CMF-pretreated and MSC control groups respectively.

 , Eight rats for normal group and 16 rats for sham operated,CMF-pretreated and MSC control groups respectively, *, EF, P = 0.03 onday 2; P = 0.001 on day 14, and FS, P = 0.01 on day 2 and P = 0.002 onday 14.

IV. Direct Therapeutic Effects in MI Model without Pre-treating MSCs andTransplantation

Referring to FIG. 5, following direct local injection of CMF in Mlmodel, it was found two weeks post infarction that in the control group(without C:MF treatment) the myocardium on the distal part of theligation site became substantially white on visual inspection due toischemic necrosis (FIG. 5: 2). By contrast, the equivalent part inCMF-treated hearts were relatively red in appearance probably due toneovascularization (FIG. 5: 1), which was comparable to the non-ischemicparts of the heart and the sizes of infarct were significantly smallerthan those in the control hearts (FIG. 5: 2). Moreover, on transect ofthe infarct area, the left ventricle walls of the CMF treated hearts(FIG. 5: 3) were significantly thicker than those in control hearts(FIG. 5: 4). Histological observations revealed that the infarct sizesin CMF-treated hearts (n=8) were on average approximately 113−½ timessmaller than those in the control hearts (n=8), as was calculated bymeasuring the infarct volume in the left ventricular free wall on day 14after ligation. By Masson's Trichrome staining, it was found that in CMFtreated hearts, myocyte-like cell clusters, which were arranged inalmost the same orientation as the infarcted myocardium or theneighboring viable myocardia, were distributed in most parts of thewhole infarct regions (FIG. 5: 5). By contrast, the infarcted regions incontrol hearts were almost completely occupied by fibrous tissuereplacement, leaving almost no space for any possible cardiomyocytesregeneration, if any (FIG. 5: 6). Under a higher power field, in a sharpcontrast to the overall blue stained fibrous scar in control group (FIG.5: 8), the whole infarct areas in the CMF treated group were filled withregenerating myocyte clusters, well shaped to bear myocardial morphologywith little fibrous tissue in between (FIG. 5: 7), although the sizes ofthese regenerating myocytes were smaller than the neighboringpreexisting myocytes, probably because they were still on the way ofmaturing.

Furthermore, echocardiography demonstrated that the replacement ofinfarcted heart tissue with structurally integrated regeneratingmyocardia and reconstituted vasculatures was accompanied by significantfunctional improvement by day 2 post-infarct in CMF-treated hearts, andfurther improvement by day 14 compared with control hearts, probably dueto the growth and maturation of the regenerated myocardia andvasculatures that repaired the infarct.

V. Therapeutic Effect via conditioned medium induced by CMF-treated MSCs

To determine whether conditioned medium containing certain inducedproteins secreted by CMF-activated-MSCs would bring about similareffects as CMF direct application or transplantation of theCMF-pretreated-MSCs to the infarct area, the conditioned medium wastested with both MSCs culture and heart infarction animal model.Referring to FIG. 6a , after treatment with conditioned medium for 24hrs, the proliferation rate of MSCs increased to 120% compared tocontrol medium (fresh growth medium). Referring to FIG. 6b , localinjection of conditioned medium to the ischemic region of MI model,cardiomyocyte regeneration was observed. Briefly, many regeneratingcardiomyocytes and many newly formed vessels filled with blood cellswere observed in the whole infarct zone (FIG. 6 b: 2) compared with thefibrous replacement in control (FIG. 6b : 1).

VI. Cellular Origin of the Regenerated Myocardia after Direct CMFTreatment

Referring to FIG. 7, studies on MI model with bone marrow replacementwith DiI labeled MSCs have provided direct evidence that the cellularorigin of the regenerating myocardium were derived from bone marrowMSCs. One week after bone marrow replacement, heart infarction surgerywas performed as described in the above. Fourteen days post infarction,it was found that the whole infarct regions in CMF-treated hearts werewell occupied by DiI labeled cells, which were largely absent in anynon-infarcted regions of having the preexisting viable cardiomyocytes.These DiI positive cells were clustered together bearing myocardial-likemorphology, but smaller in size compared with pre-existingcardiomyocytes (FIG. 7: 1). By contrast, only a few scattered DiIpositive cells were observed along the infarct border zone in controlinfarcted hearts (FIG. 7: 2). To confirm that these well organized DiIpositive cells were regenerating myocardia, immunohistochemistry withantibodies specific to troponin I and PCNA were performed. It was foundthat numerous DiI positive cells distributed in the whole infarct zonewere positively stained by specific antibodies for troponin I (FIG. 7:3) or PCNA (FIG. 7: 4). These DiI and troponin I or PCNA positivelystained cells were organized into myocardial-like tissue in the wholeinfarct zone in CMF treated hearts (FIGS. 7: 3 & 4). Under higher powerfield, these regenerating myocardial-like tissues showed the typicalmorphology of myocardium with clear intercalated disk connection betweenregenerating myocytes, indicating the ultrastructural maturation of theindividual regenerating myocyte into integrated myocardium (FIG. 7: 5).Without the structural integration between the regenerating myocytes,and between the regenerating myocardium and pre-existing viablemyocardium, functional integration and synchronous mechanical activitywould not be guaranteed. These regenerating myocardia occupied averagely69.3% of the total infarct volume on day 14 post-infarct. By contrast,in 6 control hearts, only a few cells were both troponin I and Dilpositive and were scattered around the vessels while the infarction zonewas mainly occupied by fibrous scar (FIG. 7: 6). These resultsdemonstrated that the CMF induced regenerating myocardium was functionaland derived from bone marrow MSCs.

VII. Manufacturing Pharmaceutical Compositions and Their Uses inTreating ischemic heart diseases in Mammals

Once the effective chemical compound is identified and partially orsubstantially pure preparations of the compound are obtained either byisolating the compound from natural resources such as plants or bychemical synthesis, various pharmaceutical compositions or formulationscan be fabricated from partially or substantially pure compound usingexisting processes or future developed processes in the industry.Specific processes of making pharmaceutical formulations and dosageforms (including, but not limited to, tablet, capsule, injection, syrup)from chemical compounds are not part of the invention and people ofordinary skill in the art of the pharmaceutical industry are capable ofapplying one or more processes established in the industry to thepractice of the present invention. Alternatively, people of ordinaryskill in the art may modify the existing conventional processes tobetter suit the compounds of the present invention. For example, thepatent or patent application databases provided at USPTO officialwebsite contain rich resources concerning making pharmaceuticalformulations and products from effective chemical compounds. Anotheruseful source of information is Handbook of Pharmaceutical ManufacturingFormulations, edited by Sarfaraz K. Niazi and sold by Culinary &Hospitality Industry Publications Services.

As used in the instant specification and claims, the term “plantextract” means a mixture of natural occurring compounds obtained fromparts of a plant, where at least 10% of the total dried mass isunidentified compounds. In other words, a plant extract does notencompass an identified compound substantially purified from the plant.The term “pharmaceutical excipient” means an ingredient contained in adrug formulation that is not a medicinally active constituent. The term“an effective amount” refers to the amount that is sufficient to elicita therapeutic effect on the treated subject. Effective amount will vary,as recognized by those skilled in the art, depending on the types ofdiseases treated, route of administration, excipient usage, and thepossibility of co-usage with other therapeutic treatment. A personskilled in the art may determine an effective amount under a particularsituation.

While there have been described and pointed out fundamental novelfeatures of the invention as applied to a preferred embodiment thereof,it will be understood that various omissions and substitutions andchanges, in the form and details of the embodiments illustrated, may bemade by those skilled in the art without departing from the spirit ofthe invention. The invention is not limited by the embodiments describedabove which are presented as examples only but can be modified invarious ways within the scope of protection defined by the appendedpatent claims.

REFERENCES

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3. Tomita, S. et al. Autologous transplantation of bone marrow cellsimproves damaged heart function. Circulation 100, 11247-56 (1999).

4. Beltrami, A. P. et al. Evidence that human cardiac myocytes divideafter myocardial infarction. N Engl J Med 344, 1750-7 (2001).

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6. Laugwitz, K. L. et al. Postnatal islI+ cardioblasts enter fullydifferentiated cardiomyocyte lineages. Nature 433, 647-53 (2005).

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8. Poss, K. D., Wilson, L. G. & Keating, M. T. Heart regeneration inzebrafish. Science 298, 2188-90 (2002).

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1. A composition comprising substantially purified compounds of thefollowing formulae:

and pharmaceutically acceptable salts thereof.
 2. The composition ofclaim 1, wherein the composition is a pharmaceutical compositioncomprising a pharmaceutically excipient.
 3. The pharmaceuticalcomposition of claim 2, wherein the composition is formulated into adosage form.
 4. The pharmaceutical composition of claim 3, wherein thedosage form is selected from the group consisting of tablet, capsule,injection solution, syrup, suspension and powder.